Exploitation of a Novel Binding Pocket in Human Lipoprotein

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Exploitation of a Novel Binding Pocket in Human Lipoprotein-Associated Phospholipase A2 (Lp-PLA) Discovered Through X-Ray Fragment Screening 2

Alison J.-A. Woolford, Joseph E. Pero, Sridhar Aravapalli, Valerio Berdini, Joseph E. Coyle, Philip J. Day, Andrew M. Dodson, Pascal Grondin, Finn P. Holding, Lydia Y. W. Lee, Peng Li, Eric S. Manas, Joseph P. Marino, Agnes C. L. Martin, Brent W McCleland, Rachel L. McMenamin, Christopher W Murray, Christopher E Neipp, Lee W. Page, Vipulkumar K. Patel, Florent Potvain, Sharna J. Rich, Ralph A. Rivero, Kirsten Smith, Donald O. Somers, Lionel Trottet, Ranganadh Velagaleti, Glyn Williams, and Ren Xie J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00212 • Publication Date (Web): 11 May 2016 Downloaded from http://pubs.acs.org on May 15, 2016

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Journal of Medicinal Chemistry

Velagaleti, Ranganadh; GlaxoSmithKline, Medicinal Chemistry Williams, Glyn; Astex Pharmaceuticals, Biophysics Xie, Ren; GlaxoSmithKline, Medicinal Chemistry

ACS Paragon Plus Environment


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Exploitation of a Novel Binding Pocket in Human Lipoprotein-Associated Phospholipase A2 (Lp-PLA2) Discovered Through X-Ray Fragment Screening

Alison J.-A. Woolford,*,† Joseph E. Pero,*,§ Sridhar Aravapalli,§ Valerio Berdini,† Joseph E. Coyle,† Philip J. Day,† Andrew M. Dodson,§ Pascal Grondin,∥ Finn P. Holding,† Lydia Y. W. Lee,† Peng Li,§ Eric S. Manas,⊥ Joseph Marino Jr,⊥ Agnes C. L. Martin,† Brent W. McCleland,§ Rachel L. McMenamin,† Christopher W. Murray,† Christopher E. Neipp,§ Lee W. Page,† Vipulkumar K. Patel,‡ Florent Potvain,∥ Sharna Rich,† Ralph A. Rivero,§ Kirsten Smith,† Donald O. Somers,‡ Lionel Trottet,∥ Ranganadh Velagaleti,§ Glyn Williams,† and Ren Xie.§

†

Astex Pharmaceuticals, 436 Cambridge Science Park, Milton Road, Cambridge CB4 0QA,

United Kingdom ‡

GlaxoSmithKline, Gunnels Wood Road, Stevenage, SG1 2NY, United Kingdom

⊥GlaxoSmithKline, §

1250 South Collegeville Road, Collegeville, PA 19426, USA

GlaxoSmithKline, 709 Swedeland Road, King of Prussia, PA 19406, USA

∥GlaxoSmithKline,

Centre de Recherches Francois Hyafil, 25-27 avenue du Québec, Les Ulis,

France *Contributed equally to the work

ABSTRACT: Elevated levels of human lipoprotein-associated phospholipase A2 (Lp-PLA2) are associated with cardiovascular disease and dementia. A fragment screen was conducted against Lp-PLA2 in order to identify novel inhibitors. Multiple fragment hits were observed in different regions of the active site, including some hits that bound in a pocket created by


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movement of a protein sidechain (approximately 13 Å from the catalytic residue Ser273). Using structure guided design we optimized a fragment that bound in this pocket to generate a novel low nanomolar chemotype, which did not interact with the catalytic residues.

KEYWORDS: Lp-PLA2, novel fragment pocket, fragment-based lead discovery, structure guided optimization.

INTRODUCTION Lipoprotein-Associated Phospholipase A2 (Lp-PLA2) is a member of the superfamily of phospholipases A2 (PLA2) that plays a key role in lipid metabolism by catalysing the hydrolysis of the sn-2 ester bond in phospholipids.1 Lp-PLA2 localises in the plasma and is associated with both low-density (~80%) and high density (~20%) lipoproteins.2 Elevated levels of circulating Lp-PLA2 are implicated in inflammation which is triggered by the hydrolysis of oxidatively damaged phospholipid substrates to form pro-inflammatory stimuli such as lysophosphatidylcholine and oxidized non-esterified fatty acids.2 A positive correlation with Lp-PLA2 levels has been found in certain disease states, including cardiovascular disorders,3 dementia,4 diabetic macular edema5 and prostate cancer.6 Consequently, inhibitors of Lp-PLA2 have been clinically investigated in atherosclerosis7 and Alzheimer’s disease.8 The first generation inhibitor of Lp-PLA2 to enter clinical trials was darapladib (1), which was derived from a high throughput screen.9 Darapladib is an orally bioavailable inhibitor, despite possessing a high molecular weight and high lipophilicity.10 Unfortunately, darapladib failed in two phase III trials primarily because it did not meet the primary endpoints for efficacy.



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Darapladib (1) MW 667, clogP11 8.3

In the primary in vitro assay (a fluorescence intensity assay),12 darapladib (1) is a potent inhibitor of Lp-PLA2 with an IC50 = 0.049 nM, ligand efficiency (LE)13 = 0.30 (Table 3). A secondary assay is used to assess non-specific binding in plasma and approximates the physiological environment of the enzyme. This assay measures the inhibition of Lp-PLA2 in whole human plasma, and darapladib shows a large drop off to an IC50 = 35 nM (LE = 0.22), presumably due to the high lipophilicity of the ligand.

The binding site of Lp-PLA2 has a deep canyon-like pocket containing a catalytic triad comprised of Ser273, His351 and Asp296, and an oxyanion hole formed by the backbone NH groups of Leu153 and Phe274 (Figure 1).14 The X-ray crystal structure of the apo protein reveals the presence of a water molecule in the oxyanion hole. The function of the oxyanion hole is to stabilise the incipient negative charge of the transition state during ester hydrolysis of a substrate.


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Figure 1. The active site Lp-PLA2 showing the catalytic triad (Ser273, His351 and Asp296) and the oxyanion hole (backbone NH groups of Leu153 and Phe274). The hydrogen bond interactions are shown as dotted lines and a water molecule as a gray sphere.

The binding mode of darapladib (1) is shown in Figure 2.15 The pyrimidinone carbonyl of 1 displaces the water and forms two hydrogen bonds in the oxyanion hole, whilst the fluorophenyl moiety sits in a deep hydrophobic pocket formed by the side chains of residues Leu107, Leu159, Ala355 and Phe357. The biphenyl makes an intramolecular edge-to-face πstacking interaction with the fluorophenyl ring and extends into a predominantly lipophilic channel towards Phe125. The highly lipophilic nature of the pocket occupied by the three phenyl rings of 1 suggests that the design of less lipophilic motifs may be challenging.

Other inhibitors of Lp-PLA2 described in the medicinal chemistry literature are also based upon the pyrimidinone core,16 as well as alternative chemotypes such carbamates,17 βlactams,18 oximes19 and xanthurenic acid.20 The pyrimidinone-based inhibitors are more advanced and possess physicochemical properties which are more in line with the rule of 5 (Ro5), but still retain a lipophilic bis-aryl ether motif.



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a

b

Figure 2. (a) Space filling X-ray crystal structure of the active site of Lp-PLA2 in complex with darapladib (1) (showing the Connolly surfaces). The hydrogen bond interactions are shown as dotted lines. Selected residues are shown. (b) A rotated view of 1 in complex with Lp-PLA2.

PLA2-VIIB is a closely related intracellular phospholipase,1a,14 which shares 42% overall sequence identity with Lp-PLA2. The binding sites of Lp-PLA2 and PLA2-VIIB are highly conserved but there are a number of differences, including Phe110 (Tyr65 in PLA2-VIIB), Gln352 (Arg315 in PLA2-VIIB), Ala355 (Thr318 in PLA2-VIIB) and Leu371 (Thr336 in PLA2-VIIB). These residues could be targeted for the purpose of generating selective LpPLA2 inhibitors. Darapladib (1) possesses a 1200-fold selectivity for Lp-PLA2 over PLA2VIIB (Table 3), presumably through its interaction with these four key selectivity residues. The toxicological consequences of PLA2-VIIB inhibition have not been determined and it was considered desirable to retain a selectivity of >100-fold.


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Our goal was to design a novel inhibitor with comparable potency (in plasma) and selectivity profile to darapladib (1), but with improved physicochemical properties. We reasoned that a less lipophilic inhibitor would help to address the poor translation between the Lp-PLA2 biochemical assay and the plasma assay. In addition, lower lipophilicity inhibitors (clogP 1mM), such as X-ray crystallography, NMR, ITC and SPR. Typically, structural information is gained during the screening phase which can then be used to drive the efficient development of a fragment into a lead molecule.

In this paper, we describe the output from a crystallographic fragment screen against LpPLA2, where we identified multiple fragment hits that occupied different regions of the active site. A subset of these fragments was observed to bind in a previously unknown pocket, formed by the reorientation of the Phe357 side chain. We demonstrate how it was possible to exploit this newly identified pocket to inhibit Lp-PLA2 without interacting with the active site residues.

RESULTS AND DICSUSSION Fragment Screen and Hits. An Astex Pyramid fragment screen24 was conducted versus LpPLA2, and comprised thermal shift, NMR and X-ray crystallographic screening arms.



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Although the crystal structure of Lp-PLA2 was published,14 we developed a novel crystal system that facilitated fragment soaking and rapid in-house data collection (see Supporting Information). A core fragment set (CFS), comprised of 1360 compounds, was simultaneously screened in thermal shift and ligand-detected NMR (LD-NMR); a 150 compound sub-set of CFS was screened as singletons in X-ray crystallography. During the NMR screen, control spectra from fragment cocktails dissolved in buffer and detergent were subtracted from LDNMR spectra of samples containing Lp-PLA2, in order to eliminate any contributions from fragment binding to detergent. In a second step, LD-NMR data were recollected after addition of a saturating concentration of a tool compound which binds at the darapladib site, allowing the total LD-NMR signal obtained for each fragment to be divided into contributions from competitive and non-competitive binding to Lp-PLA2. Any fragment hits derived from thermal shift or LD-NMR were only pursued if they could be validated in an X-ray crystal structure. From this process a total of 34 X-ray validated hits were obtained. Substructure searching around the X-ray validated hits identified a number of commercially available and in-house compounds that were subjected to virtual screening using the docking program GOLD25. This generated a further 16 X-ray validated hits.

The binding site and the superimposed structures of representative fragment hits 2-6 are shown in Figure 3. The fragments bind to different regions in the active site of Lp-PLA2 and together define a binding surface comparable to that exhibited by darapladib (1). Typically, fragments bind to distinct regions (“hot spots”) within a binding site and so the contiguous fragment binding in the Lp-PLA2 active site was an unexpected observation. This observation may indicate that the binding affinity of ligands such as 1 is the result of multiple binding intertactions partitioned over the surface of the ligand, and that this may reflect the extended size and lipophilicity of lipid substrates.


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Figure 3. Superposition of the X-ray crystal structures of the representative fragment hits 2-6 (2 in cyan, 3 in magenta, 4 in dark blue, 5 in green and 6 in orange), that bind throughout the active site of Lp-PLA2 (showing the Connolly surfaces). Selected residues are shown.



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(a) Fragment 2 O

(b) Fragment 3

N N O

(d) Fragment 5

O

O N

(c) Fragment 4

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NH

(e) Fragment 6

O F

H 2N

N

H2N

S O

O

O

Cl

Figure 4a-e. The X-ray crystal structures of fragments 2-6 in Lp-PLA2; 2 (cyan) adjacent to the catalytic residues Ser273, His351 and Asp296; 3 (magenta) bound in the oxyanion hole; 4 (dark blue); 5 (green); and 6 (orange) in the Lp-PLA2 “Phe357 moved” conformation. The hydrogen bond interactions are shown as dotted lines and water molecules as gray spheres. Selected residues are shown.

A more detailed diagram of the binding modes of fragments 2-5 is shown in Figure 4a-d. Fragment 2 (IC50 = ~1 mM)26 binds via a face-to-face π-stacking interaction with the indolyl of Trp298 and forms a hydrogen bond between the carbonyl and a water molecule occupying the oxyanion hole (Figure 4a). The isobutyl tail of fragment 2 points towards a solvent exposed region and is disordered in the electron density. Hydantoin 3 (IC50 >1 mM)26 binds in the oxyanion hole (Figure 4b). The urea carbonyl of hydantoin 3 forms two hydrogen bonds with the backbone NH of both Leu153 and Phe274, and recapitulates the interactions formed by the pyrimidone carbonyl in 1. In contrast, sulfonamide 4 (IC50 >1 mM,26 ITC Kd = 3.4 mM) does not form any direct hydrogen bonds with the oxyanion hole (Figure 4c), but forms two hydrogen bonds to the backbone carbonyl of Leu153 and the side chain of Gln352. The phenyl ring occupies a deep lipophilic pocket that overlaps closely with the space occupied by


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the fluorophenyl moiety of 1. Phe357 is approximately 13 Å from the Cα of the catalytic residue Ser273 and forms another binding site for lipophilic fragments, such as amide 5 (IC50 = ~500 µM,26 ITC Kd = 980 µM). The aryl ring in amide 5 forms an edge-to-face π-stack with the phenyl ring of Phe357 (Figure 4d). The ligand carbonyl forms a single water-mediated hydrogen bond to the carbonyl of Leu369 and the alkene undergoes a π-stacking interaction with the phenyl of Phe110.

A subset of fragments, such as bis-aryl 6 (IC50 = ~100 µM,12,26 LE = ~0.36), bound in an novel pocket that had not been previously observed in Lp-PLA2 crystal structures (Figure 4e). This new pocket was generated by a -22° rotation of the Cα-Cβ bond in Phe357 and results in a ~3 Å displacement of the C-4 in the phenyl side chain from its position in the apo-protein structure. An overlay of the binding modes of amide 5 and bis-aryl 6 illustrates the rotation of the Phe357 side chain (Figure 5a). The aromatic ring of Phe357 forms an edge-to-face π– stacking interaction with both of the aryl rings of the twisted bis-aryl 6. The aniline ring of 6 is in van der Waals contact with the side chains of Leu111 and Leu371, whilst the chlorophenyl ring inserts into the new pocket and forms π–stacking interactions with a number of aromatic rings including Phe125 and Phe357. An analysis of this and other fragments indicated that a dihedral angle of ~90 degrees was required for the binding of the hydrophobic bis-aryl motif.



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a

b

Figure 5. (a) Superposition of the X-ray crystal structures of amide 5 (orange) and bis-aryl 6 (gray) in complex with Lp-PLA2. The ligand Connolly surface is shown. Movement of the phenyl ring of Phe357 (black arrow) generates the fragment pocket. The hydrogen bond interactions are shown as dotted lines and water as orange or gray spheres. Only selected residues are shown. (b) X-ray crystal structure of amide 8 bound to Lp-PLA2.

Identification of this novel pocket provided an opportunity to design novel Lp-PLA2 inhibitors that were not dependent on interactions with the oxyanion hole and might possess improved physicochemical properties. Although the clogP of fragment 6 is high (clogP 3.5), bis-aryls with a large dihedral angle tend to have a higher solubility due to the minimization of intermolecular π-stacking between the aromatic rings in the crystal lattice.27 The conformationally restricted geometry is favoured due to the ortho substitution on each ring and this pre-organization probably enhances the binding affinities of these fragments.28 Crucially, fragment 6 possessed a suitable growth vector from the nitrogen to an area of the protein where polar interactions were observed (i.e. with the carbonyl of Leu153 and the sidechain of Gln352) in other fragment hits.


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Fragment Growing from Bis-Aryl 6. Overlay of the X-ray structures of 5 and 6 (Figure 5a) led to the design of the hybrid amide 7. Amide 7 had sub-micromolar affinity for Lp-PLA2 (IC50 = 180 nM) and an excellent LE = 0.44 (Table 1). We introduced pyridine nitrogens to each ring in order to lower the clogP of the bis-aryl template but the polarity was not tolerated (data not shown). However, we did achieve a modest decrease in clogP by substituting the chloro (7) for a methyl (8), and this compound binds as expected to Lp-PLA2 (Figure 5b).

Pyrroline 8 Re-Design. Concerns about the chemical stability of the pyrroline ring in 8 led us to explore replacement heterocycles. Introducing saturated heterocycles such as the pyrrolidine 9 (IC50 = 30 µM) caused a surprising and unacceptable 130 fold decrease in affinity. Direct exchange of the pyrroline (8) for a thiazole (10) provided a more acceptable 9fold loss in potency. We also explored replacing the ketone with an NH because the anilino moiety was tolerated in fragment 6. The resulting aminothiazole 11 was observed to be equipotent to ketothiazole 10. The X-ray structures unambiguously identified the thiazole sulfur atom in 10 and 11, and revealed that the thiazole rings were rotated 180 degrees relative to one another (Figure 6). The ketothiazole conformational preference is driven by the attractive carbonyl-sulfur interaction, whereas the aminothiazole conformation is driven by an intramolecular hydrogen bond between the thiazole N and the linker NH.29 Thus, further growth from the thiazoles 10 and 11 would require different regioisomer substitutions. We chose to focus on the aminothiazole moiety for two reasons. Firstly, the aminothiazole offered a superior growth vector from the C-4; and secondly, the conformation would be easier to replicate with non-sulfur containing heterocycles in future designs. At this stage the analogues 10 and 11 did not show any selectivity over PLA2-VIIB (Table 1).



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Table 1. Structure-Based Optimization of Bis-Aryl 6

rhLp-PLA2 IC50

rhPLA2-VIIB

(µM)a / LE

IC50 (µM)b / LE

3.5

~100 / ~0.36

43 / 0.40

Cl

3.5

0.18 / 0.44

>10

8

Me

3.2

0.23 / 0.43

89 / 0.26

9

Me

3.5

30 / 0.29

>100 / -

10

Me

4.6

2.0 / 0.37

3.2 / 0.36

11

Me

5.3

1.7 / 0.39

11 / 0.34

Cpd

R1

R2

clogP

6

Cl

NH2

7

All assay details are described in Supporting Information a

Recombinant human Lp-PLA2 Thio-PAF assay

b

Recombinant human PLA2-VIIB Thio-PAF assay


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a

b

Figure 6. X-ray crystal structures of (a) ketothiazole 10, and (b) aminothiazole 11, bound to the Lp-PLA2 “Phe357 moved” conformation. The sulfur atoms were clearly identifiable from the electron density (not shown) and unambiguously confirmed the orientation of the thiazoles. Hydrogen bond interactions are shown as dotted lines and water molecules as gray spheres. Arrows show a growth vector.

Growth of Thiazole 11. The X-ray structure of aminothiazole 11 (Figure 6b) shows the three aromatic rings forming multiple lipophilic interactions with the protein. The structure also reveals a growth vector into a groove adjacent to Gln352. This groove is a narrow channel which contains polar groups such as the carbonyl of Leu153 and the primary amide of Gln352, and is occupied by two water molecules. We reasoned that targeting these residues by addition of a small polar group to the thiazole C-4 could simultaneously lower the clogP of the template and increase selectivity (Gln352 is substituted by Arg315 in PLA2-VIIB). The hydroxypropyl thiazole 12 was designed to occupy this channel and chosen for synthesis (Table 2). Thiazole 12 showed an increase in Lp-PLA2 inhibition (IC50 = 230 nM) and selectivity (>40-fold selective versus PLA2-VIIB). The binding mode of thiazole 12 (Figure 7a) provided a possible explanation for the improved selectivity because a subtle reorientation



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of the side chain of Gln352 facilitated the formation of a new hydrogen bond to the nitrogen of the thiazole.


Cpd

R3

clogP

rhLp-PLA2 IC50 (µM)a / LE

rhPLA2-VIIB IC50 (µM)b / LE

Plasma IC50 (µM)c / LE

11

H

5.3

1.7 / 0.39

11 / 0.34

-

12

4.9

0.23 / 0.38

>10

7.1 / 0.29

(±)-13

4.4

0.0095 / 0.42

>10

0.56 / 0.33



b

c


Lp-PLA2 in whole human plasma Thio-PAF assay

It was also noted that thiazole 12 did not access a small lipophilic sub-pocket located between the side chains of Tyr160 and Phe357 (Figure 7a); the bottom of the pocket is partially formed by the sidechain of Ala355 which is substituted by a larger threonine residue in PLA2-VIIB. We therefore introduced a methoxy group from C-2 of the n-propyl in thiazole 12 to give


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hydroxy ether in 13. Thiazole 13 had an IC50 of 9.5 nM indicating the addition of the methoxy (2 heavy atoms) had resulted in a 24-fold increase in inhibitory potency due to improved shape complementarity with the sub-pocket. This represents an excellent group efficiency30 of 0.95 for the methoxy group. In line with our expectations, thiazole 13 showed improved selectivity over PLA2-VIIB (>1000-fold).

Table 2 also shows the potency of thiazoles 12 and 13 against Lp-PLA2 in a whole human plasma assay. Both compounds show a significant drop off in activity in the plasma assay and are still some way off our goal of reaching comparable plasma potency to darapladib of IC50 = 35 nM (Table 3).



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a

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b H 2N O

C-6

HN

HN N

S

N N O OH

OH

12

16

Figure 7. (a) The X-ray crystal structure of thiazole 12 in Lp-PLA2 in the “Phe357 moved” conformation (showing the Connolly surfaces). The circle highlights an unfilled space. The C6 position of the central aryl ring of 12 is solvent exposed. The hydrogen bond interactions are depicted as dotted lines and a hydrogen bond is shown between the thiazole nitrogen and Gln352. Water molecules are shown as gray spheres. (b) The X-ray crystal structure of pyrazole 16 in Lp-PLA2 in the “Phe357 moved” conformation (showing the Connolly surfaces).


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Elaboration of Thiazole 13. In order to further improve the potency of thiazole 13 (clogP 4.4) in the Lp-PLA2 human plasma assay, we sought to modulate the physicochemical properties, and reduce the lipophilicity of the template. Exchanging the thiazole for more polar heterocycles such as triazole or oxadiazole failed to retain potency (data not shown) and our strategy was modified to introduce a polar group to the C-6 position on the central aryl ring (depicted in Figure 7a). The central phenyl ring is in contact with the side chains of Leu111 and Leu371, and the C-6 vector points towards solvent. An ether linker was chosen to retain a co-planar arrangement with the central aryl ring, whilst permitting growth towards the bulk solvent region. Methoxy analogue 14 and aminoethoxy analogue 15 both demonstrated comparable potency to parent thiazole 13 and retained good selectivity over PLA2-VIIB (Table 3). More interestingly, the reduced lipophilicity of aminoethoxy 15 (clogP 3.6) resulted in an 11-fold increase in the plasma assay potency (IC50 = 28 nM).

With previous SAR indicating that the thiazole nitrogen was important to activity and that polarity in other parts of the thiazole ring was poorly tolerated, we investigated a pyrazole as a potential replacement. Pyrazole 16 was equipotent to thiazole 15 in both the Lp-PLA2 biochemical and plasma assays, and retained selectivity over PLA2-VIIB.

The binding pose of pyrazole 16 is shown in Figure 7b where the (S)-isomer has been preferentially selected. The orientation of the hydroxylmethyl moiety is inferred from the hydrogen bonds formed to the NH2 of the Gln352 and to a water molecule; the methoxy fills the small hydrophobic pocket beside Ala355. A second direct hydrogen bond between pyrazole 16 and the protein is between the terminal amine of 16 and the Leu369 carbonyl. There are also a number of water mediated contacts with a network of waters across the top of the pocket.



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R4

MW / clogP

rhLp-PLA2 IC50 (µM)a / LE

rhPLA2VIIB IC50 (µM)b / LE

Plasma IC50 (µM)c / LE

-H

369 / 4.4

0.0095 / 0.42

>10

0.56 / 0.33

(±)-14

399 / 4.4

0.0036 / 0.41

6.1 / 0.25

0.32 / 0.32

(±)-15

428 / 3.6

0.0024 / 0.39

>10

0.028 / 0.34

(±)-16

411 / 3.4

0.0014 / 0.40

>1

0.018 / 0.35

667 / 8.3

0.000049 / 0.30

0.063 / 0.21

0.035 / 0.22

Cpd

Heterocycle

(±)-13

1

Darapladib



b

c


Lp-PLA2 in whole human plasma Thio-PAF assay


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Table 3 also shows that we have achieved our initial goals of attaining comparable potency (in plasma) and selectivity to darapladib (1), and reduced the ratio between the Lp-PLA2 biochemical assay and the plasma assay. Darapladib has a ratio of 700; whereas both thiazole 15 and pyrazole 16 have only a 12 fold difference.

Table 4 shows additional properties of thiazole 15 and pyrazole 16. These compounds both have a molecular weight less than 500 Da, and a clogP of 3.6 and 3.4, respectively. The difference in liophilicity between the thiazole and the pyrazole is quite small but is supported by measurements of ChromLogDpH

7.4

for these compounds (3.4 and 3.2 respectively). In

contrast, darapladib showed a 2 unit disconnect between the clogP = 8.3 and ChromLogDpH 7.4 = 6.2.

Table 4 also shows that thiazole 15 and pyrazole 16 have comparable human plasma protein binding and artificial membrane permeability to darapladib (1), but aqueous solubility is vastly improved presumably due the lower lipophilicity of the chemotype.

The data for our lead compounds readily satisfied our physiochemical property goals, despite the fragment originating from a highly lipophilic pocket, and compares favourably to LpPLA2 inhibitors in the clinic and from the medicinal chemistry literature.16-20



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Table 4. In Vitro Data

MW / clogP

ChromLogD at pH 7.4

Aq. Solubility (µM)

hPPB

Permeability (nm/sec)

(±)-15

428 / 3.6

3.4

195

97%

210

(±)-16

411 / 3.4

3.2

302

95%

130

667 / 8.3

6.2

8

98%

230

Cpd

1

Hetero cycle

R4

Darapladib

Synthesis of Pyrazole 16. Synthesis of pyrazole 16 is displayed in Schemes 1-3. Aminopyrazole intermediate 20 was prepared in four steps (Scheme 1). Treatment of 5-nitro1H-pyrazole with ethyl oxirane-2-carboxylate under basic conditions yielded pyrazole 17. The alcohol was subsequently methylated with methyl iodide in the presence of silver(I) oxide to afford nitro pyrazole 18. Selective reduction of the nitro group with iron powder gave amine 19, and finally reduction of the ester afforded the aminopyrazole intermediate 20.


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Scheme 1. Synthesis of Intermediate 20a

17

19 a

18

20

Reagents and conditions: (a) K2CO3, DMF, rt, 18 h, 25%; (b) MeI, Ag2O, MeCN, 40 ºC, 18

h, 63%; (c) Fe, NH4Cl, H2O, EtOH, 70 °C, 3 h, 86%; (d) LiBH4, THF, rt, 2 h, 41%.

Iodo intermediate 24 was prepared in five steps (Scheme 2). A palladium-catalysed cross coupling reaction between 2-methylphenylboronic acid and 1-bromo-4-fluoro-2-methyl-5nitrobenzene gave bis-aryl 21 in high yield. Treatment of bis-aryl 21 with N-(2hydroxyethyl)phthalimide and NaH initiated an SNAr reaction to afford aryl ether 22. The nitro moiety was reduced with hydrogen over Pd/C to yield aniline 23, which was subjected to a one pot diazotization−iodination cascade in the presence of NaNO2, KI and p-TsOH to give iodo intermediate 24.



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Scheme 2. Synthesis of Intermediate 24a

21

22

23

24 a

Reagents and conditions: (a) Pd(PPh3)4, Cs2CO3, 1,4-dioxane, H2O, 85 ºC, 4 h, 91%; (b)

NaH, THF, rt, 5 h, 73%; (c) H2, 10% Pd/C, MeOH, rt, 5 h, 70%; (d) KI, NaNO2, p-TsOH, MeCN, H2O, rt, 3 h, 20%.

The final coupling is shown in Scheme 3 where phthalimide 25 was prepared by a palladium assisted cross-coupling of aminopyrazole intermediate 20 and iodo intermediate 24. Finally, removal of the phthalimide with hydrazine yielded the desired pyrazole 16.


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Scheme 3. Synthesis of Pyrazole 16a

20

24

25

16 a

Reagents and conditions: (a) Pd(OAc)2, Xantphos, Cs2CO3, 1,4-dioxane, 100 ºC, 3 h; (b)

NH2NH2, EtOH, rt, 2 h, 8% two steps.

CONCLUSION In summary, we have used a fragment-based approach to identify the first potent Lp-PLA2 inhibitors which do not make a direct interaction with the catalytic residues of the enzyme. Fragment screening of Lp-PLA2 led to the identification of numerous fragment hits that effectively mapped the active site of the enzyme and occupied a binding surface similar to that defined by darapladib (1). A sub-set of fragment hits were revealed to bind in a novel pocket, approximately 13 Å from the oxyanion hole, formed by rotation of the Phe357 side



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chain. Among these was bis-aryl fragment 6, which was subsequently optimized using structure based principles and grown into regions of the active site highlighted by other hits from the fragment screen. Although fragment growth began from a very lipophilic region of the protein pocket, the optimization process led to the efficient design of a potent and selective chemotype with respectable physicochemical properties despite containing this bisaryl motif (i.e. pyrazole 16: MW 411, ChromLogDpH 7.4 3.4, solubility 302 µM). By carefully controlling the physicochemical properties of these inhibitors, we were able to identify leading exemplars (i.e. thiazole 15 and pyrazole 16) which exhibited a lower drop off between the Lp-PLA2 biochemical and plasma assays relative to darapladib. Ultimately, work was halted in this series because neither thiazole 15 nor pyrazole 16 possessed PK properties consistent with once-daily dosing in humans.31 Nonetheless, this work highlights the success of fragment based drug discovery in identifying ligand efficient compounds with vastly improved physicochemical properties. Future publications will describe other fragment derived chemotypes with attractive physicochemical properties and PK profiles supportive of further advancement.

EXPERIMENTAL SECTION Chemistry. General directions are described in the Supporting Information. The synthesis of pyrazole 16 is described below. A full description of the synthetic protocols and chemical characterizations for compounds 2-15 can be found in the Supporting Information. The purity of each compound was analysed by HPLC−MS (ESI) and is >95%, unless otherwise stated.

Procedures.

3-(3-{[2-(2-Aminoethoxy)-4-methyl-5-(2-methylphenyl)phenyl]amino}-1H-

pyrazol-1-yl)-2-methoxypropan-1-ol trifluoroacetic acid salt (16). A mixture of 3-(3amino-1H-pyrazol-1-yl)-2-methoxypropan-1-ol (20) (71.6 mg, 0.418 mmol), 2-{2-[2-iodo-5-


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methyl-4-(2-methylphenyl)phenoxy]ethyl}-2,3-dihydro-1H-isoindole-1,3-dione (24) (160 mg, 0.322 mmol), Pd(OAc)2 (14 mg, 0.062 mmol), Xantphos (55 mg, 0.095 mmol) and Cs2CO3 (314 mg, 0.965 mmol) in 1,4-dioxane (3.0 mL) was heated to 100 °C (microwave) for 3 hours. After allowing to cool to ambient temperature, the mixture was filtered through a bed of Celite® and the filtrate was partitioned between water and EtOAc. The organic layer was washed with brine and dried over MgSO4. After filtration, the solvent was evaporated in vacuo and the crude product was purified by flash chromatography (gradient elution with 080% EtOAc in hexanes) to yield 2-[2-(2-{[1-(3-hydroxy-2-methoxypropyl)-1H-pyrazol-3yl]amino}-5-methyl-4-(2-methylphenyl)phenoxy)ethyl]-2,3-dihydro-1H-isoindole-1,3-dione (25) (44 mg). LCMS (method 3): m/z 541.1 [M+H]+, RT = 1.22 min. The residue was taken up in EtOH (1.5 mL) and hydrazine hydrate (12 µL, 0.382 mmol) was added. The solution was stirred for 2 hours at ambient temperature. The mixture was loaded onto a SCX cartridge, and washed sequentially with methanol and 2.0 M NH3 in methanol solution. The NH3 solution was collected and evaporated in vacuo. The product was purified by preparative HPLC to yield 3-(3-{[2-(2-aminoethoxy)-4-methyl-5-(2-methylphenyl)phenyl]amino}-1Hpyrazol-1-yl)-2-methoxypropan-1-ol trifluoroacetic acid salt (16) as an off-white solid (11 mg, 8% two steps). 1H NMR (CDCl3): δ 8.64 (br s, 3H), 7.46 (s, 1H), 7.27-7.24 (m, 3H), 7.12 (d, J = 8.0 Hz, 1H), 7.03 (s, 1H), 6.82 (s, 1H), 6.16 (s, 1H), 4.39-4.23 (m, 4H), 3.71-3.57 (m, 6H), 3.40 (s, 3H), 2.10 (s, 3H), 2.04 (s, 3H).

13

C NMR (CD3OD): δ 151.5, 144.2, 141.2,

135.4, 134.6, 131.6, 130.8, 129.1 129.0, 126.5, 125.6, 125.0, 115.2, 112.4, 94.9, 80.5, 64.5, 60.5, 56.6, 51.8, 38.9, 18.4, 17.8. LCMS (method 3): m/z 411.2 [M+H]+, RT = 0.86 min. Purity >95%. HRMS: Found 411.2385, C23H30N4O3 [M+H]+ requires 411.2391 (∆ = -1.43 ppm). X-ray crystal structure gained in Lp-PLA2.



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Ethyl 2-hydroxy-3-(3-nitro-1H-pyrazol-1-yl)propanoate (17). 3-Nitro-1H-pyrazole (4.9 g, 43.3 mmol), K2CO3 (12.0 g, 86.7 mmol) and ethyl oxirane-2-carboxylate (5.0 g, 43.3 mmol) in DMF (24 mL) were stirred at ambient temperature for 18 hours. The reaction mixture was diluted with water and the product extracted with EtOAc (x3). The combined organic layers were dried over Na2SO4, filtered and evaporated in vacuo. The product was purified by flash chromatography (gradient elution with 0-40% EtOAc in petrol) to afford ethyl 2-hydroxy-3(3-nitro-1H-pyrazol-1-yl)propanoate (17) as a colourless oil (2.5 g, 25%). 1H NMR (DMSOd6): δ 7.98 (d, J = 2.5 Hz, 1H), 7.03 (d, J = 2.5 Hz, 1H), 6.00 (br s, 1H), 4.55-4.47 (m, 2H), 4.43 (dd, J = 15.1, 8.7 Hz, 1H), 4.20-4.08 (m, 2H), 1.20 (t, J = 7.1 Hz, 3H).

13

C NMR

(DMSO-d6): δ 171.1, 155.1, 135.2, 102.5, 69.1, 60.7, 55.8, 13.9. LCMS (method 2): m/z 229.9 [M+H]+, RT = 1.17 min. Purity 90%. HRMS: Found 230.0771, C8H11N3O5 [M+H]+ requires 230.0772 (∆ = -0.31 ppm).

Ethyl 2-methoxy-3-(3-nitro-1H-pyrazol-1-yl)propanoate (18). To a stirred solution of ethyl 2-hydroxy-3-(3-nitro-1H-pyrazol-1-yl)propanoate (17) (180 mg, 0.79 mmol) in MeCN (3 mL) was added Ag2O (274 mg, 1.18 mmol) and MeI (0.147 mL, 2.36 mmol). The mixture was stirred in Reacti-VialTM at 40 °C for 18 hours. The reaction mixture was filtered through a bed of Celite® and washed with EtOAc. The filtrate was evaporated in vacuo to afford crude compound. Purification of the product was performed by flash chromatography (gradient elution with 0-40% EtOAc in petrol) to afford ethyl 2-methoxy-3-(3-nitro-1H-pyrazol-1yl)propanoate (18) as a colourless oil (121 mg, 63%). 1H NMR (CDCl3): δ 7.57 (d, J = 2.5 Hz, 1H), 6.89 (d, J = 2.5 Hz, 1H), 4.61 (dd, J = 14.0, 3.4 Hz, 1H), 4.40 (dd, J = 14.0, 8.0 Hz, 1H), 4.31-4.23 (m, 2H), 4.21 (dd, J = 8.0, 3.4 Hz, 1H), 3.43 (s, 3H), 1.32 (t, J = 7.1 Hz, 3H). 13

C NMR (CDCl3): δ 169.2, 156.3, 134.0, 103.0, 79.0, 62.0, 59.2, 55.3, 14.3. LCMS (method


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2): m/z 243.8 [M+H]+, RT = 1.27 min. Purity >95%. HRMS: Found 244.0932, C9H13N3O5 [M+H]+ requires 244.0928 (∆ = 1.69 ppm).

Ethyl 3-(3-amino-1H-pyrazol-1-yl)-2-methoxypropanoate (19). To a solution of ethyl 2methoxy-3-(3-nitro-1H-pyrazol-1-yl)propanoate (18) (121 mg, 0.50 mmol), in water (2 mL) and EtOH (5 mL), was added iron powder (139 mg, 2.49 mmol) and NH4Cl (266 mg, 4.98 mmol). The reaction mixture was stirred at 70 °C for 3 hours, and then allowed to cool to ambient temperature. The reaction was diluted with sat. NaHCO3 (aq.) and extracted with EtOAc (x3). The combined organic phases were dried with MgSO4, filtered and evaporated in vacuo to afford ethyl 3-(3-amino-1H-pyrazol-1-yl)-2-methoxypropanoate (19) as a colourless oil (92 mg, 86%). 1H NMR (CDCl3): δ 7.17-7.15 (m, 1H), 5.57-5.54 (m, 1H), 4.29-4.24 (m, 1H), 4.21 (q, J = 7.1 Hz, 2H), 4.12-4.03 (m, 2H), 3.62 (br s, 2H), 3.35 (s, 3H), 1.27 (t, J = 7.1 Hz, 3H). 13C NMR (CDCl3): δ 170.4, 154.8, 132.2, 93.4, 79.8, 61.5, 59.0, 53.6, 14.3. LCMS (method 2): m/z 214.0 [M+H]+, RT = 1.08 min. Purity >95%. HRMS: Found 214.1193, C9H15N3O3 [M+H]+ requires 214.1186 (∆ = 3.21 ppm).

3-(3-Amino-1H-pyrazol-1-yl)-2-methoxypropan-1-ol (20). To a stirred solution of ethyl 3(3-amino-1H-pyrazol-1-yl)-2-methoxypropanoate (19) (92 mg, 0.43 mmol) in THF (2 mL) was added lithium borohydride (19 mg, 0.86 mmol). The reaction mixture was stirred for 2 hours at ambient temperature. The mixture was treated with water (2 mL) and the resulting precipitate was filtered. The filtrate was evaporated in vacuo and the residue taken up in MeOH:water (1:1, 1 mL). The product was directly purified by preparative HPLC to yield 3(3-amino-1H-pyrazol-1-yl)-2-methoxypropan-1-ol (20) as a clear colourless oil (31 mg, 41%). 1H NMR (CDCl3): δ 7.18 (d, J = 2.3 Hz, 1H), 5.59 (d, J = 2.3 Hz, 1H), 4.16-4.01 (m,



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2H), 3.75-3.55 (m, 4H), 3.55-3.48 (m, 1H), 3.38 (s, 3H), 3.09 (br s, 1H). 13C NMR (CDCl3): δ 154.7, 132.2, 93.1, 80.2, 61.2, 57.9, 51.7. LCMS (method 2): m/z 172.2 [M+H]+, RT = 0.44 min. Purity >95%. HRMS: Found 172.1087, C7H13N3O2 [M+H]+ requires 172.1081 (∆ = 3.52 ppm).

4-Fluoro-2,2'-dimethyl-5-nitro-1,1'-biphenyl

(21).

1-Bromo-4-fluoro-2-methyl-5-

nitrobenzene (2.00 g, 8.55 mmol), 2-methylphenylboronic acid (1.39 g, 10.24 mmol), Cs2CO3 (3.06 g, 9.40 mmol) were taken up in 1,4-dioxane:water (5:1, 28.5 mL). The reaction was degassed with nitrogen for 10 minutes. Pd(PPh3)4 (200 mg, 0.171 mmol) was added and the reaction was stirred at 85 ºC for 4 hours. The solvent was evaporated in vacuo and the residue was partitioned between water and EtOAc. The aqueous layer was separated and extracted further with EtOAc (x2). The combined organic layers were washed with brine, dried over MgSO4, filtered and evaporated in vacuo. The product was purified by flash chromatography (gradient elution with 0-6% EtOAc/petrol) to yield 4-fluoro-2,2'-dimethyl-5-nitro-1,1'biphenyl (21) as a pale yellow solid (1.92 g, 91%). 1H NMR (DMSO-d6): δ 7.81 (d, J = 7.8 Hz, 1H), 7.60 (d, J = 12.5 Hz, 1H), 7.37-7.33 (m, 2H), 7.32-7.26 (m, 1H), 7.13 (d, J = 7.4 Hz, 1H), 2.10 (s, 3H), 2.02 (s, 3H). 13C NMR (DMSO-d6): δ 153.5 (d, J = 260.4 Hz), 146.3 (d, J = 8.8 Hz), 137.9 (d, J = 3.7 Hz), 137.6, 135.3, 134.4 (d, J = 8.1 Hz), 130.1, 129.0, 128.3, 126.1 (d, J = 2.2 Hz), 126.0, 119.3 (d, J = 20.5 Hz), 19.7, 19.3. LCMS (method 2): m/z 263.0 [M+NH4]+, RT = 1.53 min. Purity >95%.

2-{2-[5-Methyl-4-(2-methylphenyl)-2-nitrophenoxy]ethyl}-2,3-dihydro-1H-isoindole-1,3dione (22). To a mixture of 4-fluoro-2,2'-dimethyl-5-nitro-1,1'-biphenyl (21) (1.64 g mg, 6.69 mmol) and 2-(2-hydroxyethyl)isoindoline-1,3-dione (1.55 g, 8.02 mmol) in THF (33 mL) was


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added NaH (290 mg, 7.36 mmol) in portions. The reaction mixture was stirred for 5 hours at ambient temperature and then quenched with 5% citric acid (aq.). The product was extracted with EtOAc (x3) and the combined organic layers were washed with brine and dried over MgSO4. After filtration, the solvent was evaporated in vacuo and the product was purified by flash chromatography (gradient elution with 0-40% EtOAc in petrol) to yield 2-{2-[5-methyl4-(2-methylphenyl)-2-nitrophenoxy]ethyl}-2,3-dihydro-1H-isoindole-1,3-dione

(22) as a

yellow foam (2.04 g, 73%). 1H NMR (CDCl3): δ 7.92-7.87 (m, 2H), 7.76-7.71 (m, 2H), 7.62 (s, 1H), 7.30-7.26 (m, 1H), 7.26-7.18 (m, 2H), 7.03 (d, J = 7.4 Hz, 1H), 6.99 (s, 1H), 4.43 (t, J = 5.9 Hz, 2H), 4.20 (t, J = 5.9 Hz, 2H), 2.08 (s, 3H), 2.02 (s, 3H). 13C NMR (CDCl3): δ 168.2, 150.8, 144.0, 138.8, 137.7, 136.1, 134.9, 134.2, 132.2, 130.3, 129.6, 128.1, 126.8, 126.0, 123.6, 116.3, 66.2, 37.0, 20.6, 19.9. LCMS (method 2): m/z 434.0 [M+NH4]+, RT = 1.52 min. Purity >95%. HRMS: Found 417.1452, C24H20N2O5 [M+H]+ requires 417.1445 (∆ = 1.54 ppm).

2-{2-[2-Amino-5-methyl-4-(2-methylphenyl)phenoxy]ethyl}-2,3-dihydro-1H-isoindole1,3-dione (23). 2-{2-[5-Methyl-4-(2-methylphenyl)-2-nitrophenoxy]ethyl}-2,3-dihydro-1Hisoindole-1,3-dione (22) (350 mg, 0.84 mmol) and 10% Pd/C (90 mg, 0.084 mmol) in MeOH (8.4 mL) was shaken for 5 hours under a hydrogen atmosphere at ambient temperature and pressure. The mixture was filtered and the filtrate was evaporated in vacuo to give 2-{2-[2amino-5-methyl-4-(2-methylphenyl)phenoxy]ethyl}-2,3-dihydro-1H-isoindole-1,3-dione (23) as a yellow solid (229 mg, 70%). 1H NMR (DMSO-d6): δ 7.93-7.89 (m, 2H), 7.89-7.85 (m, 2H), 7.26-7.13 (m, 4H), 6.97-6.94 (m, 1H), 6.68 (s, 1H), 4.44 (s, 2H), 4.18 (t, J = 5.4 Hz, 2H), 4.03 (t, J = 5.4 Hz, 2H), 1.96 (s, 3H), 1.80 (s, 3H). LCMS (method 2): m/z 387.0 [M+H]+, RT = 1.51 min. Purity >90%.



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2-{2-[2-Iodo-5-methyl-4-(2-methylphenyl)phenoxy]ethyl}-2,3-dihydro-1H-isoindole-1,3dione

(24).

To

a

stirred

solution

of

2-{2-[2-amino-5-methyl-4-(2-

methylphenyl)phenoxy]ethyl}-2,3-dihydro-1H-isoindole-1,3-dione (23) (229 mg, 0.62 mmol) in MeCN (4.1 mL) was added p-TsOH (359 mg, 1.87 mmol) at ambient temperature. A solution of NaNO2 (86 mg, 1.24 mmol) and KI (248 mg, 1.49 mmol) in water (1.2 mL) was introduced slowly. The mixture was stirred for 3 hours at ambient temperature and then diluted with water. The product was extracted with EtOAc and the organic layer was subsequently washed with water, brine and dried over MgSO4. After filtration, the solvent was evaporated in vacuo and the crude product was purified by flash chromatography (gradient elution with 0-15% EtOAc in petrol) to yield 2-{2-[2-iodo-5-methyl-4-(2methylphenyl)phenoxy]ethyl}-2,3-dihydro-1H-isoindole-1,3-dione (24) as a light brown oil (62 mg, 20%). 1H NMR (CD3OD): δ 7.92-7.88 (m, 2H), 7.84-7.81 (m, 2H), 7.35 (s, 1H), 7.26-7.17 (m, 3H), 7.01-6.97 (m, 1H), 6.92 (s, 1H), 4.40 (t, J = 5.7 Hz, 2H), 4.16 (t, J = 5.7 Hz, 2H), 2.00 (s, 3H), 1.98 (s, 3H). 13C NMR (CD3OD): δ 168.3, 156.2, 139.8, 139.3, 137.6, 136.8, 135.7, 134.0, 132.2, 129.5, 129.1, 127.2, 125.3, 122.8, 113.9, 81.6, 65.2, 36.9, 18.6, 18.5. LCMS (method 2): m/z 498.0 [M+H]+, RT = 1.68 min. Purity 90%. HRMS: Found 498.0567, C24H20INO3 [M+H]+ requires 498.0561 (∆ = -1.16 ppm).

AUTHOR INFORMATION Corresponding Authors (A.J.-A.W.) Tel: +44(0)1223 226283. Fax: +44(0)1223 226201. [email protected] (J.E.P.) Tel: +1 610 270 6515. Fax: +1 610 270 4451. [email protected]


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ACKNOWLEDGMENTS We would like to thank many colleagues including David Rees, Jeff Yon, Chris Johnson, Stuart Whibley, Torren Peakman and Marie-Hélène Fouchet for providing support during the project, and Marc O’Reilly, Jeff St Denis and Ben Cons for helpful comments on the manuscript.

ABBREVIATIONS USED ITC, isothermal titration calorimetry; %I, percent inhibition of signal relative to solvent-only control; MW, molecular weight in Daltons; petrol, Petroleum ether; p-TsOH, pToluenesulfonic acid monohydrate; SPR, Surface plasmon resonance; Xantphos, 4,5Bis(diphenylphosphino)-9,9-dimethylxanthene.

ASSOCIATED CONTENT Supporting Information Supporting Information contains biophysical assay protocols, supplementary tables, crystallographic details, PDB accession codes, computational methods and programs used, in vitro assay details, PK details, synthetic schemes, experimental procedures and characterization of organic molecules.

ACCESSION CODES Coordinates for the Lp-PLA2 complexes with compounds 2, 3, 4, 5, 6, 8, 10, 11, 12 and 16 have been deposited in the Protein Data Bank (PDB) under accession codes: 5jad (2), 5jah (3), 5jal (4), 5jan (5), 5jao (6), 5jap (8), 5jar (10), 5jas (11), 5jat (12) and 5jau (16). Authors will release the atomic coordinates in experimental data upon article publication.



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Fused

imidazopyrimidinones

as

Lp-LPA2

inhibitors


and

their

preparation.


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as

zinc-

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See supporting information for PK characterisation.


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Table of Contents graphic

Fragments bound throughout the active site of Lp-PLA2.


Exploitation of a Novel Binding Pocket in Human Lipoprotein

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